Aging in mammals can have a profound deleterious effect on brain function that manifests primarily in deficits of cognitive and motor function. Longevity genes are thus of obvious interest and importance, both for their life-extension potential and the possibility of their contributing to the enhancement of the quality of life, particularly later during the lifespan. However, very few of these genes have been identified and even less is understood about how these genes act to prevent aging and promote life extension.
Accordingly, there exists the need to discover genes whose function is associated with life-extension. Such genes and their products would be useful in the screening for anti-aging agents and would serve as key targets in various anti-aging therapies. Indeed, an understanding of the mechanisms underlying aging will ultimately provide us with the tools necessary to alleviate these deficits in the aged population and thereby prolong the independence of the elderly.
The roundworm C. elegans is a valuable invertebrate model system to study aging owing to its short, reproducible life span and its amenability to genetic and molecular analysis. Further, as the entire C. elegans genome is sequenced, it is feasible to envisage a comprehensive identification of all the genes that affect aging in this organism. In molecular genetics, extended life span remains one of the best indicators that an intervention in an aging process has been made. The life span of C. elegans is easily extendable by various genetic, transgenic, and pharmacologic means, and the isolation of long-lived variants in C. elegans has begun to provide clear insights into the putative mechanisms and consequences of aging in the CNS.
The main pathway that regulates life span in C. elegans is an insulin-like signaling pathway. Mutations in genes in this pathway can increase, decrease, or have no effect on life span. Interestingly, several of these genes were first isolated based on their effects on development. The normal lifecycle of C. elegans follows development from an egg, through four larval stages, and a final molt into a fertile, adult hermaphrodite. When nutrition is low or population density is high, the worms can undergo an alternative developmental program to form “dauer” larvae (Cassada R. C. & Russell R. (1975) Dev. Biology 46:326-342). The dauer larvae is a diapause stage that does not feed or reproduce, is stress resistant and is apparently non-aging, wherein worms can remain as dauer larvae for months (Klass M. R. & Hirsh D. I. (1976) Nature 260:523-525). When conditions improve, worms can re-enter the life cycle and develop into a normal reproductive hermaphrodite. The dauer formation genes (daf), or genes that determine the decision to progress through development normally or undergo dauer formation, were first isolated on the basis that they either promote dauer arrest under plentiful growth conditions (dauer constitutive) or prevent dauer formation under crowded conditions (dauer defective) (Riddle D. L. et al. in C. elegans II, (1997) 739-768, Cold Spring Harbor Laboratory Press). Several of these genes, including daf-2, age-1 and daft 6, were subsequently identified as part of an insulin-like signaling pathway, supporting the idea that genes that affect entry into the dauer stage also affect life span in C. elegans. 
The insulin-like signaling pathway in C. elegans contains numerous genes, many of which were isolated originally through their effects on dauer formation. Of the 37 insulin family members that have been identified in the C. elegans genome, only one insulin receptor-like gene, daf-2 (Pierce S. B. et al. (2001) Genes and Dev. 15:672-686; Gregoire F. M. et al. (1998). Biochem Biophys Res Com. 249:385-390) has been clearly identified. DAF-2 highly resembles both the mammalian insulin receptor and the related insulin growth factor-1 receptor (IGF1-R) (Kimura K. et al. (1997) Science 277:942-946). The ligand that binds to the DAF-2 receptor is not yet known. Activation of the insulin-like receptor DAF-2 by the as yet unidentified ligand leads to activation of PI-3 kinase, which in turn results in the generation of phosphoinositide-3-phosphate (PIP3). In mammalian systems, PIP3 acts as an intracellular messenger to activate downstream kinases (Kido Y. et al. (2001) J of Clin End and Met 86: 972-979; Alessi, D. R. & Downes, C. P., (1998) Biochim Biophys Acta 1436: 151-164). In C. elegans, the catalytic subunit, p110, of PI-3 kinase is encoded by the age-1 gene (Morris J. Z. et al. (1996) Nature 382:536-539). Decrease in function mutations in either daf-2 or age-1 result in various phenotypes including constitutive dauer formation during development, fertility defects, resistance to stresses such as heat, oxidative damage and heavy metals, and extension of life span in adults (Lithgow G. J. et al., (1994) J. Gerontol. 49:B270-276; Lithgow G. J. et al., (1995) PNAS USA 92:7540-4; Murakami S. & Johnson T. E. A Genetics 143:1207-1218; Honda Y. & Honda S., (1999) FASED J 13:1385-1393; Baryste D., (2001) FasEB J 15:627-634; Friedman D. B. & Johnson T. E., (1988) Genetics 118:75-86; Klass M. R., (1983) Mech of Ageing and Dev. 22:279-286). Another gene in the pathway, daf-18, encodes a homolog of the mammalian tumor suppressor PTEN phosphatase (Rouault J. P., (1999) Curr Biology 9:329-332; Ogg S. & Ruvkun G., (1998) Mol Cell 2:887-893; Mihaylova V. T. et al., (1999) PNAS USA 96:7427-7432; Gil E. B. et al. (1999) PNAS USA 96:2925-2930). DAF-18 functions to regulate the levels of PIP3 by dephosphorylating the inositol ring in the third position (Maehama T. & Dixon J. E. (1998) J Biol Chem 273:13375-13378). Loss of function mutations in daf-18 result in a decrease in life span and suppression of both daf-2 and age-1 dauer phenotypes (Rouault J. P. (1999) Curr Biol 9:329-332; Ogg S. & Ruvkun G. (1998) Mol Cell 2:887-893; Mihaylova V. T. (1999) PNAS USA 96:7427-7432; Gil E. B. et al. (1999) PNAS USA 96:2925-2930.)
Downstream of age-1 are the kinases PDK-1, AKT-1, and AKT-2. The PDK-1 and AKT-1 kinases were identified in C. elegans as gain-of-function suppressors of the dauer-constitutive phenotype of age-1 mutants (Paradis S. & Ruvkun G. (1998) Genes Dev 12:2488-2498; Paradis S. (1999) Genes Dev 13:1438-1452). Similar to the phenotype observed for mutations in daf-2 and age-1, a reduction of function mutation in PDK-1 increases adult life span (Paradis S. (1999) Genes Dev 13:1438-1452). The final output of the pathway is daf-16, which encodes a homolog of the HNF-3/forkhead family of transcription factors (Kimura K. et al. (1997) Science 277:942-946; Ogg S. et al. (1997) Nature 389:994-9; Lin K. et al. (1997) Science 278:1319-1322). Null mutations of daf-16 decrease life span and completely suppress all phenotypes in double mutant combinations with daf-2 or age-1. Thus life span extension by either daf-2 or age-1 mutations requires a wild type daf-16 gene. Given that DAF-1 and AGE-1 proteins act to suppress the activity of DAF-16, it is believed that the lack of signaling in daf-2 or age-1 mutants causes increased activity of DAF-16, ultimately leading to the observed phenotypes. The final targets of DAF-16 in this pathway remain unknown but are presumed to regulate metabolism and fat storage (Kimura K. et al. (1997) Science 277:942-946; Lithgow G. J. et al. (1995) PNAS USA 92:7540-4).
In order to elucidate fully the mechanisms underlying aging, it will be critical to identify all pathways that play a role in its regulation. Importantly, genetic analysis of the DAF-2 insulin-like receptor strongly indicates that other genes are involved in signaling downstream of daf-2 (Ogg S. & Ruvkun G. (1998) Mol Cell 2:887-893). This is due to the fact that a loss of function mutation in the daf-16 forkhead transcription factor completely suppresses all of the phenotypes of a loss of function mutation in either daf-2 or age-1 (Kenyon, C. in C. elegans II (1997) 791-813, Cold Spring Harbor Press; Tissenbaum H. A. & Ruvkun G. (1998) Genetics 148:703-717)). However, loss of function mutations in daf-18 as well as gain of function mutation in either akt-1 and pdk-1 only suppress a subset of the phenotypes associated with the daf-2 and age-1 loss of function mutations (Paradis S. & Ruvkun G. (1998) Genes Dev 12:2488-2498; Paradis S. et al. Genes Dev 13:1438-1452; Ogg S. & Ruvkun G. (1998) Mol Cell 2:887-893). These data indicate that an additional pathway(s) is active downstream of daf-2 but upstream of daf-16. There exists, therefore, a clear need in the art for the elucidation of additional pathways involved in regulating aging.
Importantly, the influence of the insulin/IGF signaling pathway on lifespan has been conserved across large evolutionary distances. In the fruit fly Drosophila, reduced insulin/IGF signaling also mediates life-span extensions (Clancy D. J. (2001) Science 292:104-106; Tatar M. & Yin C. (2001) Exp. Gerontol. 36:723-738). This conservation indicates that certain physiological processes effecting life span are very ancient and strongly suggests that information on the aging of simple animals is likely to be important for mammalian aging. The study of development and longevity in C. elegans is thus expected to uncover critical new targets for insulin regulators in higher organisms and potential anti-aging targets for drug intervention in humans.